PROJECT SUMMARY/ABSTRACT Less than 1 in 10 individuals who attempt to quit smoking remain abstinent for 1 year. This poor quit rate is driven, in part, by the fact that currently available drugs used to aid in smoking cessation are only moderately effective, at best. Thus, there is a great need to develop novel drugs that are more effective for smoking cessation. It is the goal of this project to use a novel genetic strategy to identify new biological targets for the potential development of novel smoking cessation drugs. The genetic strategy is based upon identifying modifier genes that alter nicotine responses in mice that have a null mutation in Chrna5, the gene that codes for the nicotinic receptor ?5 subunit. In effect, modifier genes are genes that contribute to physiological and/or molecular processes that are important for the behavior of interest but that generally go undetected in the absence of a perturbation in the gene that they modify. Because variants in Chrna5 alter risk for nicotine dependence in humans and studies in rodents clearly demonstrate that Chrna5 is critical for many nicotine- related behaviors, we believe that identifying genes that modify the effect of Chrna5 deletion on nicotine behaviors will uncover new genes relevant to nicotine dependence that may serve as novel targets for novel smoking cessation pharmacotherapies. Importantly, the behaviors that we plan to screen for modifiers are not only dependent upon Chrna5, but also dependent uponthe medial habenula-IPN pathway, a neural pathway that is thought to play a critical role in nicotine dependence. To identify genetic modifiers of the effect of Chrna5 deletion on nicotine behaviors, we propose 3 aims. In specific aim 1, we will breed the Chrna5 null mutation onto each of the B6-ChrA/J chromosome substitution strains (CSS) and identify chromosomes that harbor modifier genes for the effect of Chrna5 deletion on three nicotine behaviors, oral nicotine intake, somatic signs of nicotine withdrawal, and nicotine conditioned place preference. For specific aim 2, we will fine map those chromosomes that harbor modifier genes using sequential congenic strains. Typically, 3 generations of congenic strains starting from a CSS strain provides mapping resolution equivalent to that of any high resolution mapping population. Finally, in specific aim 3, we will use RNA-seq to identify genes whose expression is altered by the identified modifier genes. Importantly, we will use a state of the art genetic strategy that will allow us to examine gene expression in a neural cell population that is highly relevant to the behaviors: Chrna5 expressing cells of the interpeduncular nucleus. By combining the results of this aim with modifier loci identified through aims 1 and 2, we expect to narrow the list of potential candidate modifier genes and identify pathways specifically impacted by the modifier genes. In short, we believe that this strategy will lead to the identification of previously unknown genes and/or genetic pathways that contribute to the physiological and/or molecular processes important for the response to nicotine. These genes and/or pathways may serve as novel targets for the development of new pharmacotherapies to aid in smoking cessation.